Iron is one of several minerals required by animals as a dietary supplement. At the biochemical level, iron is complexed with and required for the normal functioning of a number of essential proteins, including hemoglobin, myoglobin, peroxidases, catalases, cytochromes, and cytochrome oxidases. Thus, among the important functions that iron/protein complexes serve are oxygen transport, detoxification, and cellular energy production in the form of ATP.
Due to the critical roles that these iron-containing proteins serve, all higher organisms express proteins responsible for the uptake, transport, and storage of iron. Upon its uptake in the small intestine, iron immediately combines with the ubiquitous serum protein transferrin, the primary vehicle by which iron is transported throughout the body. The uptake of circulating iron-transferrin complexes into cells is mediated by CD71, the transferrin receptor. Once inside the cell, iron is dissociated from transferrin and is then incorporated into newly synthesized iron-containing proteins or, if present in excess, is stored in the liver by binding with apoferritin to form the iron storage protein complex known as ferritin.
Because iron is required by the cytochrome oxidase enzyme complex to produce cellular ATP, a constant supply of iron/transferrin complexes has been determined to be an essential factor for the growth of most cultured cells (Trowbridge, Prog. Allergy, 1988, 45, 121-146). In order for cells to uptake and utilize this extracellular source of iron, the expression of functional CD71 is required. Evidence for this comes from the finding in a number of cell lines that the level of cell surface CD71 is directly correlated to proliferation rate (Neckers, Pathobiology, 1991, 59, 11-18). Further, stimulation of quiescent T- and B-lymphocytes in response to mitogens or antigen exposure has been reported to be transferrin-dependent as well (Neckers, Pathobiology, 1991, 59, 11-18). Finally, exposure of cells to anti-CD71 antibodies has been found to cause cytostasis and eventually cell death (Trowbridge, Prog. Allergy, 1988, 45, 121-146).
The requirement of both iron/transferrin complexes and CD71 for cell proliferation has led to the idea that inhibition of iron utilization could represent a novel strategy for the treatment of cancer. In support of this notion, in vitro studies have demonstrated inhibition of tumor cell growth using chelating agents that extract iron from transferrin, monoclonal antibodies that block CD71 function and gallium nitrate, a compound which blocks the dissociation of iron from transferrin within cells (Kemp, Histol. Histopathol., 1997, 12, 291-296; Trowbridge, Prog. Allergy, 1988, 45, 121-146). Studies with tumor xenografts in mice indicate that anti-tumor effects can be achieved by iron deprivation under in vivo conditions as well. For example, administration of anti-CD71 antibodies alone or in combination with the iron chelator deferoxamine has shown anti-tumor activity against lymphoid tumor growth (Kemp, Histol. Histopathol., 1997, 12, 291-296) and T-cell leukemia (White et al., Cancer Res., 1990, 50, 6295-6301). Similarly, blockage of iron utilization with gallium nitrate has displayed anti-tumor effects against both hematopoietic and solid tumors (Kemp, Histol. Histopathol., 1997, 12, 291-296).
Antisense mediated inhibition of CD71 has been utilized as a tool to investigate non-CD71 mechanisms of iron uptake into cells. Thus, Sasaki et al. disclosed an antisense oligodeoxynucleotide targeted to the transcriptional start site of CD71 mRNA (Sasaki et al., Am. J. Hematol., 1993, 42, 74-80). This oligodeoxynucleotide was reported to have a minimal effect on CD71 protein expression. Also, in that study, an expression vector containing a CD71 gene in antisense orientation downstream from the cytomegalovirus promoter was found to result in approximately 50% reduction of cell surface CD71 expression when stably expressed in transformant hepatoma cell lines.
To date, strategies aimed at inhibiting CD71 function under in vivo conditions have involved the use of various monoclonal antibodies. However, it has been reported that repeated treatment with anti-CD71 antibodies results in the outgrowth of resistant tumor cells due to the development of mutations in CD71 which alter the epitope recognized by the antibodies (Lesley and Schulte, Mol. Cell. Biol., 1984, 4, 1675-1681). There remains a long felt need for additional agents capable of effectively inhibiting CD71 function.